Bottom Line:
MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner.With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side).The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.

Background: In contrast to mammals, zebrafish successfully regenerate retinal ganglion cell (RGC) axons after optic nerve section (ONS). This difference is explained on the one hand by neurite growth inhibitors in mammals (including Nogo-A), as opposed to growth-promoting glial cells in the fish visual pathway, and on the other hand by the neuron-intrinsic properties allowing the upregulation of growth-associated proteins in fish RGCs but not in mammals.

Results: Here, we report that Rtn4b, the zebrafish homologue of mammalian Nogo-A/RTN4-A, is upregulated in axotomized zebrafish RGCs and is primarily associated with the endoplasmic reticulum (ER). Rtn4b functions as a neuron-intrinsic determinant for axon regeneration, as was shown by downregulating Rtn4b through retrogradely transported morpholinos (MOs), applied to the optic nerve at the time of ONS. MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner. With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side). Moreover, regenerating axons 7d after ONS and MO1 or MO2 application were labeled by Alexa488, applied distal to the first lesion. The number of Alexa488 labeled RGCs, containing the Rtn4b MO1 or MO2, was reduced by 54% and 62%, respectively, over control MO.

Conclusions: Thus, Rtn4b is an important neuron-intrinsic component and required for the success of axon regeneration in the zebrafish visual system. The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.

Fig4: Downregulation of Rtn4b in RGCs by MO1. After application of the control (Co) MO to the lesioned optic nerve, Rtn4b labeling of RGCs in retina whole mounts was intense at 5 days after ONS (A,B), but was markedly reduced when MO1 against Rtn4b (C,D) was offered. (B,D) The Rtn4b AB-labeled cells contain Lissamine (red) conjugated to the MOs. Examples of RGCs are outlined (white interrupted lines). DAPI stains the nuclei. Scale bar, 10 μm. (E) Western blot analysis showing a significant decrease in Rtn4b expression in retinae 5 days after ONS and application of MO1 and MO2 (P < 0.0001), respectively, to the optic nerve. Alpha-tubulin served as loading control.

Mentions:
To determine whether Rtn4b is indeed needed for RGC axon regeneration, we downregulated Rtn4b by placing a MO-soaked piece of gel foam directly at the eye-sided optic nerve at the time of ONS [17]. The MOs are retrogradely transported into the RGCs as is demonstrated by using lissamine-labeled MOs. To minimize the danger of potential off-target effects, two different MOs, MO1 and MO2, were used and applied at two different concentrations (either 70 or 140 μM) to the left optic nerve in parallel with control MO application to the right nerve within the same animal. That MO1 and MO2 specifically downregulate Rtn4b in a concentration-dependent manner was demonstrated earlier in zebrafish embryos [16]. Five days after MO1 or MO2 application, the Rtn4b immunostaining intensity in RGCs in whole mounts was markedly reduced (Figure 4C,D), whereas RGCs receiving control MO were intensively labeled (Figure 4A, B). That MO1 and MO2 led indeed to Rtn4b downregulation was confirmed by Western blots of retinae, 5 days after ONS and MO application (Figure 4E) showing an overall significant reduction of Rtn4b by 32% with each MO1 and MO2 (P < 0.0001, MO1; P < 0.0001, MO2).Figure 4

Fig4: Downregulation of Rtn4b in RGCs by MO1. After application of the control (Co) MO to the lesioned optic nerve, Rtn4b labeling of RGCs in retina whole mounts was intense at 5 days after ONS (A,B), but was markedly reduced when MO1 against Rtn4b (C,D) was offered. (B,D) The Rtn4b AB-labeled cells contain Lissamine (red) conjugated to the MOs. Examples of RGCs are outlined (white interrupted lines). DAPI stains the nuclei. Scale bar, 10 μm. (E) Western blot analysis showing a significant decrease in Rtn4b expression in retinae 5 days after ONS and application of MO1 and MO2 (P < 0.0001), respectively, to the optic nerve. Alpha-tubulin served as loading control.

Mentions:
To determine whether Rtn4b is indeed needed for RGC axon regeneration, we downregulated Rtn4b by placing a MO-soaked piece of gel foam directly at the eye-sided optic nerve at the time of ONS [17]. The MOs are retrogradely transported into the RGCs as is demonstrated by using lissamine-labeled MOs. To minimize the danger of potential off-target effects, two different MOs, MO1 and MO2, were used and applied at two different concentrations (either 70 or 140 μM) to the left optic nerve in parallel with control MO application to the right nerve within the same animal. That MO1 and MO2 specifically downregulate Rtn4b in a concentration-dependent manner was demonstrated earlier in zebrafish embryos [16]. Five days after MO1 or MO2 application, the Rtn4b immunostaining intensity in RGCs in whole mounts was markedly reduced (Figure 4C,D), whereas RGCs receiving control MO were intensively labeled (Figure 4A, B). That MO1 and MO2 led indeed to Rtn4b downregulation was confirmed by Western blots of retinae, 5 days after ONS and MO application (Figure 4E) showing an overall significant reduction of Rtn4b by 32% with each MO1 and MO2 (P < 0.0001, MO1; P < 0.0001, MO2).Figure 4

Bottom Line:
MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner.With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side).The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.

Background: In contrast to mammals, zebrafish successfully regenerate retinal ganglion cell (RGC) axons after optic nerve section (ONS). This difference is explained on the one hand by neurite growth inhibitors in mammals (including Nogo-A), as opposed to growth-promoting glial cells in the fish visual pathway, and on the other hand by the neuron-intrinsic properties allowing the upregulation of growth-associated proteins in fish RGCs but not in mammals.

Results: Here, we report that Rtn4b, the zebrafish homologue of mammalian Nogo-A/RTN4-A, is upregulated in axotomized zebrafish RGCs and is primarily associated with the endoplasmic reticulum (ER). Rtn4b functions as a neuron-intrinsic determinant for axon regeneration, as was shown by downregulating Rtn4b through retrogradely transported morpholinos (MOs), applied to the optic nerve at the time of ONS. MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner. With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side). Moreover, regenerating axons 7d after ONS and MO1 or MO2 application were labeled by Alexa488, applied distal to the first lesion. The number of Alexa488 labeled RGCs, containing the Rtn4b MO1 or MO2, was reduced by 54% and 62%, respectively, over control MO.

Conclusions: Thus, Rtn4b is an important neuron-intrinsic component and required for the success of axon regeneration in the zebrafish visual system. The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.